Untranslated Region Sequences Research Articles

Migration of Small Ribosomal Subunits on the 5' Untranslated Regions of Capped Messenger RNA.

Several control mechanisms of eukaryotic gene expression target the initiation step of mRNA translation. The canonical translation initiation pathway begins with cap-dependent attachment of the small ribosomal subunit (SSU) to the messenger ribonucleic acid (mRNA) followed by an energy-dependent, sequential ‘scanning’ of the 5′ untranslated regions (UTRs). Scanning through the 5′UTR requires the adenosine triphosphate (ATP)-dependent RNA helicase eukaryotic initiation factor (eIF) 4A and its efficiency contributes to the specific rate of protein synthesis. Thus, understanding the molecular details of the scanning mechanism remains a priority task for the field. Here, we studied the effects of inhibiting ATP-dependent translation and eIF4A in cell-free translation and reconstituted initiation reactions programmed with capped mRNAs featuring different 5′UTRs. An aptamer that blocks eIF4A in an inactive state away from mRNA inhibited translation of capped mRNA with the moderately structured β-globin sequences in the 5′UTR but not that of an mRNA with a poly(A) sequence as the 5′UTR. By contrast, the nonhydrolysable ATP analogue β,γ-imidoadenosine 5′-triphosphate (AMP-PNP) inhibited translation irrespective of the 5′UTR sequence, suggesting that complexes that contain ATP-binding proteins in their ATP-bound form can obstruct and/or actively block progression of ribosome recruitment and/or scanning on mRNA. Further, using primer extension inhibition to locate SSUs on mRNA (‘toeprinting’), we identify an SSU complex which inhibits primer extension approximately eight nucleotides upstream from the usual toeprinting stop generated by SSUs positioned over the start codon. This ‘−8 nt toeprint’ was seen with mRNA 5′UTRs of different length, sequence and structure potential. Importantly, the ‘−8 nt toeprint’ was strongly stimulated by the presence of the cap on the mRNA, as well as the presence of eIFs 4F, 4A/4B and ATP, implying active scanning. We assembled cell-free translation reactions with capped mRNA featuring an extended 5′UTR and used cycloheximide to arrest elongating ribosomes at the start codon. Impeding scanning through the 5′UTR in this system with elevated magnesium and AMP-PNP (similar to the toeprinting conditions), we visualised assemblies consisting of several SSUs together with one full ribosome by electron microscopy, suggesting direct detection of scanning intermediates. Collectively, our data provide additional biochemical, molecular and physical evidence to underpin the scanning model of translation initiation in eukaryotes.

Genetic constructs creation using Golden Gate cloning method

Aim. Creation of genetic constructions to study the effects of various regulatory elements, namely promoters, on the expression of GFP reporter protein. Methods. For creation genetic constructs, the method of molecular cloning Golden Gate was used, which allows the rapid creation of genetic vectors using IIS type restriction enzymes and T4 DNA liga-ses. Results. For research six different promoters were selected, namely the 35S CaMV (Cauliflower Mosaic Virus), double 35S CaMV promoter, promoters of the RbcS2B and RbcS1B genes encoding a small subunit of ribulozobisphosphate carboxylase (RuBisCo) isolated from Arabidopsis thaliana (L.) Heynh.; promoters of genes encoding chlorophyll a-b binding proteins (LHB1B1 and LHB1B2) also isolated from A. thaliana (L.) Heynh. All transcription units additionally contained the following elements: the 5'-untranslated region Ω sequence (5’UTR Ω) from the tobacco mosaic virus TMV (Tobacco Mosaic Virus); the coding sequence of the gene gfp (Green Fluorescent Protein) isolated from A. victoria and the 35S Terminator CaMV with the polyadenylation signal and the 3'-untranslated region sequence. As a result, six genetic constructs with different regulatory elements, namely promoters, have been created. Conclusions. To study the effects of various regulatory elements, namely promoters, on the expression of a GFP repor-ter protein in transient or stable genetic transformation of plants the created genetic constructs can be used.Keywords: cloning, genetic constructs, promoters, Green Fluorescent Protein (GFP).

Up-regulation of miR-381 inhibits NAD+ salvage pathway and promotes apoptosis in breast cancer cells.

Nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme involved in nicotinamide adenine dinucleotide (NAD) salvage pathway, is overexpressed in many human malignancies such as breast cancer. This enzyme plays a critical role in survival and growth of cancer cells. MicroRNAs (miRNAs) are among the most important regulators of gene expression, and serve as potential targets for diagnosis, prognosis, and therapy of breast cancer. Therefore, the aim of this study was to assess the effect of NAMPT inhibition by miR-381 on breast cancer cell survival. MCF-7 and MDA-MB-231 cancer cell lines were transfected with miR-381 mimic, inhibitor, and their corresponding negative controls (NCs). Subsequently, the level of NAMPT and NAD was assessed using real-time PCR, immuno-blotting, and enzymatic methods, respectively. In order to evaluate apoptosis, cells were labelled with Annexin V-FITC and propidium iodide and analyzed by flow cytometry. Bioinformatics analysis was performed to recognize whether NAMPT 3′-untranslated region (UTR) is a direct target of miR-381 and the results were authenticated by the luciferase reporter assay using a vector containing the 3′-UTR sequence of NAMPT. Our results revealed that the 3′-UTR of NAMPT was a direct target of miR-381 and its up-regulation decreased NAMPT gene and protein expression, leading to a notable reduction in intracellular NAD and subsequently cell survival and induction of apoptosis. It can be concluded that miR-381 has a vital role in tumor suppression by down-regulation of NAMPT, and it can be a promising candidate for breast cancer therapy.

Human 5' UTR design and variant effect prediction from a massively parallel translation assay.

The ability to predict the impact of cis-regulatory sequences on gene expression would facilitate discovery in fundamental and applied biology. Here we combine polysome profiling of a library of 280,000 randomized 5' untranslated regions (UTRs) with deep learning to build a predictive model that relates human 5' UTR sequence to translation. Together with a genetic algorithm, we use the model to engineer new 5' UTRs that accurately direct specified levels of ribosome loading, providing the ability to tune sequences for optimal protein expression. We show that the same approach can be extended to chemically modified RNA, an important feature for applications in mRNA therapeutics and synthetic biology. We test 35,212 truncated human 5' UTRs and 3,577 naturally occurring variants and show that the model predicts ribosome loading of these sequences. Finally, we provide evidence of 45 single-nucleotide variants (SNVs) associated with human diseases that substantially change ribosome loading and thus may represent a molecular basis for disease.

NEW INSIGHT INTO THE MECHANISM OF ACTION OF ε-ELEMENT ENHANCER IN E. COLI

Epsilon element (ε-element) is a complex translational enhancer capable of promoting the prokaryotic translation efficiency in combination with the Shine-Dalgarno (SD) sequence. However, the detailed mechanism of action of ε-element is unknown, and there is no single hypothesis to explain the available evidence. We here propose a new hypothesis for the mechanism of action of ε-element from the 5′-untranslated region (UTR) of the 10 gene of phage T7 during translation initiation in Escherichia coli. The mechanism of action was considered to be driven by the complementary interaction of ε-element (and its variation εII) with the 3′-end of E. coli 16S rRNA, specifically with the 45th hairpin region. This hypothesis was then applied to currently available data of the influence of ε-element on protein expression in bacterial cells. Several 5′-UTR sequences were constructed containing variations of ε-element, and their properties were predicted according to this new hypothesis and confirmed by measuring the protein expression level. We further applied temperature stresses (cold and heat shock) to these variants to examine their influence on protein expression controlled by various enhancers. Overall, the εII_SD and ε_SD enhancers could significantly increase the protein expression level in E. coli cells under cold shock, representing a 6–9-times increase relative to that obtained with the classical SD sequence.

U13 → C13 mutation in the variable region of the NA gene 3′UTR of H9N2 influenza virus influences the replication and transcription of NA and enhances virus infectivity

The untranslated regions within viral segments are the essential promoter elements required for the initiation of viral replication and transcription. The end of the UTR sequence and part of the ORF sequence constitute the packaging signal for progeny viruses. To explore the influence of single-point and multi-site joint mutations in the UTR of the NA gene on the viral expression, we select clones with upregulated expression of the reporter gene and analyze their sequence characteristics. Bioinformatics methods were used to analyze polymorphisms in the untranslated region (UTR) of the neuraminidase gene of the H9N2 influenza A virus. Using the RNA polymerase I reporting system with enhanced green fluorescence protein (EGFP) gene as the reporter gene, libraries containing random mutations at sites within the N2 UTR were constructed using random mutagenesis. The mutants were selected from the randomized mutagenesis libraries for the N2-UTR. The N2-UTR-RNA polymerase I fluorescence reporter system was identified by sequencing and transfected into infected MDCK cells. The expression of the reporter EGFP was observed using fluorescence microscopy, and the relative fluorescence intensity was measured using a multifunctional microplate reader to analyze the expression of the reporter gene (EGFP) qualitatively and quantitatively. Herein, an RNA polymerase reporter system was constructed to rescue the mutated viruses and measure their tissue culture infective dose (TCID50). The results showed that the U13 → C13 mutation in the 3'end of the NA gene promoted the expression of viral RNA and protein, and mutation of other sites within the UTR could differentially regulate viral genomic transcription and translation. These data showed that the U13 → C13 mutation within the variable region of the 3'UTR of the NA gene in the H9N2 influenza virus promotes viral genomic expression and infection.

Discovery of Small-Molecule Antibiotics against a Unique tRNA-Mediated Regulation of Transcription in Gram-Positive Bacteria.

The emergence of multidrug-resistant bacteria necessitates the identification of unique targets of intervention and compounds that inhibit their function. Gram-positive bacteria use a well-conserved tRNA-responsive transcriptional regulatory element in mRNAs, known as the T-box, to regulate the transcription of multiple operons that control amino acid metabolism. T-box regulatory elements are found only in the 5'-untranslated region (UTR) of mRNAs of Gram-positive bacteria, not Gram-negative bacteria or the human host. Using the structure of the 5'UTR sequence of the Bacillus subtilis tyrosyl-tRNA synthetase mRNA T-box as a model, in silico docking of 305 000 small compounds initially yielded 700 as potential binders that could inhibit the binding of the tRNA ligand. A single family of compounds inhibited the growth of Gram-positive bacteria, but not Gram-negative bacteria, including drug-resistant clinical isolates at minimum inhibitory concentrations (MIC 16-64 μg mL-1 ). Resistance developed at an extremely low mutational frequency (1.21×10-10 ). At 4 μg mL-1 , the parent compound PKZ18 significantly inhibited in vivo transcription of glycyl-tRNA synthetase mRNA. PKZ18 also inhibited in vivo translation of the S. aureus threonyl-tRNA synthetase protein. PKZ18 bound to the Specifier Loop in vitro (Kd ≈24 μm). Its core chemistry necessary for antibacterial activity has been identified. These findings support the T-box regulatory mechanism as a new target for antibiotic discovery that may impede the emergence of resistance.

Occult hepatitis C virus infection in patients with beta-thalassemia major: Is it a neglected and unexplained phenomenon?

Occult hepatitis C virus (HCV) infection (OCI) is described as the presence of viral genome in both hepatocytes and peripheral blood mononuclear cells (PBMCs) despite constant negative results on serum HCV RNA tests. Beta-thalassemia major (BTM) describes a group of inherited blood diseases. Patients with BTM require repeated blood transfusions, increasing the risk of exposure to infectious agents. We aimed to assess the prevalence of OCI in Iranian BTM patients and to identify the role of host factors in OCI positivity. A total of 181 BTM patients with HCV negative markers were selected. HCV RNA was tested in PBMCs using nested polymerase chain reaction assay. The positive samples were then genotyped via restriction fragment-length polymorphism (RFLP) and 5'-untranslated region sequencing. Six (3.3%) out of 181 BTM patients had viral HCV genomes in PBMC samples. Three (50.0%), two (33.3%), and one (16.7%) out of these six patients were infected with HCV-1b, HCV-1a, and HCV-3a, respectively. OCI positivity was significantly associated with the serum level of uric acid (P = 0.045) and ABO blood group (P = 0.032). Also, OCI patients had unfavorable IFNL3 rs12979860 TT, IFNL3 rs8099917 GG, IFNL3 rs12980275 GG, and IFNL4 ss469415590 ∆G/∆G genotypes. In conclusion, we indicated the low frequency of OCI in BTM patients. Nevertheless, more attention is warranted considering the importance of this infection. Also, further studies are necessary to determine the actual prevalence of OCI among BTM patients in Iran.

Stabilizing role of structural elements within the 5´ Untranslated Region (UTR) and gag sequences in Mason-Pfizer monkey virus (MPMV) genomic RNA packaging

ABSTRACT The Mason-Pfizer monkey virus (MPMV) genomic RNA (gRNA) packaging signal is a highly-structured element with several stem-loops held together by two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences. These LRIs play a critical role in maintaining the structure of the 5´ end of the MPMV gRNA. Thus, one could hypothesize that the overall RNA secondary structure of this region is further architecturally held together by three other stem loops (SL3, Gag SL1, and Gag SL2) comprising of sequences from the distal parts of the 5´untranslated region (5ʹ UTR) to ~ 120 nucleotides into gag, excluding gag sequences involved in forming the U5-Gag LRIs. To provide functional evidence for the biological significance of these stem loops during gRNA encapsidation, these structural motifs were mutated and their effects on MPMV RNA packaging and propagation were tested in a single round trans-complementation assay. The mutant RNA structures were further studied by high throughput SHAPE (hSHAPE) assay. Our results reveal that sequences involved in forming these three stem loops do not play crucial roles at an individual level during MPMV gRNA packaging or propagation. Further structure–function analysis indicates that the U5-Gag LRIs have a more important architectural role in stabilizing the higher order structure of the 5´ UTR than the three stem loops which have a more secondary and perhaps indirect role in stabilizing the overall RNA secondary structure of the region. Our work provides a better understanding of the molecular interactions that take place during MPMV gRNA packaging.

Interrogation of Eukaryotic Stop Codon Readthrough Signals by in Vitro RNA Selection.

RNA signals located downstream of stop codons in eukaryotic mRNAs can stimulate high levels of translational readthrough by the ribosome, thereby giving rise to functionally distinct C-terminally extended protein products. Although many readthrough events have been previously discovered in Nature, a broader description of the stimulatory RNA signals would help to identify new reprogramming events in eukaryotic genes and provide insights into the molecular mechanisms of readthrough. Here, we explore the RNA reprogramming landscape by performing in vitro translation selections to enrich RNA readthrough signals de novo from a starting randomized library comprising >1013 unique sequence variants. Selection products were characterized using high-throughput sequencing, from which we identified primary sequence and secondary structure readthrough features. The activities of readthrough signals, including three novel sequence motifs, were confirmed in cellular reporter assays. Then, we used machine learning and our HTS data to predict readthrough activity from human 3'-untranslated region sequences. This led to the discovery of >1.5% readthrough in four human genes (CDKN2B, LEPROTL1, PVRL3, and SFTA2). Together, our results provide valuable insights into RNA-mediated translation reprogramming, offer tools for readthrough discovery in eukaryotic genes, and present new opportunities to explore the biological consequences of stop codon readthrough in humans.

MiR663a‑TTC22V1 axis inhibits colon cancer metastasis.

An increasing number of studies have demonstrated that microRNAs (miRs) may act as oncogenes or anti-oncogenes in various types of cancer, including colon cancer (CC). However, the clinical and biological significance of miR663a in the prognosis of CC and its underlying molecular mechanisms remain unknown. Using the reverse transcription-quantitative polymerase chain reaction on CC and surgical margin tissue samples from 172 patients with CC, it was identified that miR663a was significantly downregulated in CC (P<0.001), particularly in metastatic CC (P=0.044). miR663a overexpression inhibited the proliferation and migration/invasion of CC cells in vitro, and also tumor growth and metastasis of CC cells in vivo. Additionally, miR663a target genes were analyzed. Inverse changes in tetratricopeptide repeat domain 22 variant 1 (TTC22V1) in response to alterations in miR663a expression were observed. miR663a decreased the reporter activity of the wild-type TTC22V1−3′ untranslated region (UTR), but did not decrease that of a 3′UTR mutant. miR663a completely abolished cell migration/invasion induced by TTC22V1 containing the wild-type 3′UTR sequence, but not that induced by TTC22V1 containing the 3′UTR mutant. An inverse correlation between miR663a and TTC22 mRNA levels was observed in CC tissues. These results suggest that TTC22V1 mRNA is a crucial miR663a target that directly promotes cell migration/invasion. TTC22, which, to the best of our knowledge, has rarely been investigated, is located in the nuclei of epithelial cells in colon stem cell niches at crypt bases, and is significantly downregulated in CC, particularly in non-metastatic CC. High TTC22V1 expression is a significant poor survival factor for patients with CC. Collectively, the results of the present study suggested that TTC22V1 may be a metastasis-associated gene and that the miR663a-TTC22V1 axis inhibited CC metastasis.

MicroRNA‑9‑5p downregulates Klf4 and influences the progression of hepatocellular carcinoma via the AKT signaling pathway.

Krüppel-like factor 4 (Klf4) is a transcriptional factor involved in the progression of hepatocellular carcinoma (HCC). However, the underlying regulatory mechanisms associated with the Klf4 gene as a tumor suppressor in HCC remain unclear. microRNAs (miRNAs or miRs) are a series of small non-coding RNAs that serve a vital role in regulating gene expression via their influence on protein translation and the associated degradation of mRNA. The mRNA expression levels of the miRNA, miR-9-5p, and Klf4 were measured using reverse transcription-quantitative polymerase chain reaction. The protein expression levels of Klf4, protein kinase B (AKT), phosphorylated (p-)AKT, mechanistic target of rapamycin (mTOR), p-mTOR, B cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) were determined by western blot analysis. Dual luciferase reporter assay was used to confirm a direct interaction between miR-9-5p and the 3′-untranslated region (3′-UTR) sequence of Klf4. Cell counting kit-8 assay, wound healing assay, Transwell migration assay and flow cytometric analysis were performed to evaluate the proliferative, migratory and apoptotic capabilities of the HCC cells. In the present study, miR-9-5p was revealed to be overexpressed in HCC as a novel upstream gene of Klf4. miR-9-5p expression was inversely associated with Klf4 expression in clinical samples. Additionally, Kaplan-Meier analysis revealed a markedly poor prognosis of HCC in the miR-9-5p high-expression group. Bioinformatics analysis revealed that miR-9-5p bound directly to the 3′-UTR of Klf4, which reduced the expression levels of Klf4. The miR-9-5p/Klf4 axis promoted HCC proliferation and migration, and inhibited HCC apoptosis. Furthermore, miR-9-5p upregulated the Bcl-2/Bax protein ratio and activated AKT/mTOR signaling. Taken together, these data demonstrated that the miR-9-5p/Klf4 axis was able to promote HCC progression, which may occur via regulation of the AKT signaling pathway, highlighting a potential novel target in HCC treatment.

Shrimp miR-1000 Functions in Antiviral Immunity by Simultaneously Triggering the Degradation of Two Viral mRNAs.

MicroRNAs (miRNAs) function as crucial suppressors of gene expression via translational repression or direct mRNA degradation. However, the mechanism of multi-gene regulation by a host miRNA in antiviral immunity has not been extensively explored. In this study, the regulation of two white spot syndrome virus (WSSV) genes by its host (Marsupenaeus japonicus shrimp) miRNA (shrimp miR-1000) was characterized. The miRNA target gene prediction showed that only two virus genes (wsv191 and wsv407) might be the targets of miR-1000. The results of insect cell transfection assays revealed that shrimp miR-1000 could target multiple virus genes (wsv191 and wsv407). The mRNA degradation analysis and RNA FISH (fluorescence in situ hybridization) analysis indicated that miR-1000 triggered the mRNA degradation of target genes through 5′-3′ exonucleolytic digestion in vivo and thereby inhibited the virus infection in shrimp. The miRNA-mediated 5′-3′ exonucleolytic digestion of target mRNAs stopped near the 3′UTR (3′untranslated region) sequence complementary to the seed sequence of miR-1000. Therefore, our study provided novel insights into how a host miRNA targeted multiple viral genes and prevented host from virus infection.

Human astroviruses: in silico analysis of the untranslated region and putative binding sites of cellular proteins.

Human astrovirus (HAstV) constitutes a major cause of acute gastroenteritis in children. The viral 5′ and 3′ untranslated regions (UTR) have been involved in the regulation of several molecular mechanisms. However, in astrovirues have been less characterized. Here, we analyzed the secondary structures of the 5′ and 3′ UTR of HAstV, as well as their putative target sites that might be recognized by cellular factors. To our knowledge, this is the first bioinformatic analysis that predicts the HAstV 5′ UTR secondary structure. The analysis showed that both the UTR sequence and secondary structure are highly conserved in all HAstVs analyzed, suggesting their regulatory role of viral activities. Notably, the UTRs of HAstVs contain putative binding sites for the serine/arginine-rich factors SRSF2, SRSF5, SRSF6, SRSF3, and the multifunctional hnRNPE2 protein. More importantly, putative binding sites for PTB were localized in single-stranded RNA sequences, while hnRNPE2 sites were localized in double-stranded sequence of the HAstV 5′ and 3′ UTR structures. These analyses suggest that the combination of SRSF proteins, hnRNPE2 and PTB described here could be involved in the maintenance of the secondary structure of the HAstVs, possibly allowing the recruitment of the replication complex that selects and recruits viral RNA replication templates.Electronic supplementary materialThe online version of this article (10.1007/s11033-018-4498-8) contains supplementary material, which is available to authorized users.

Circular RNA CDR1as regulates osteoblastic differentiation of periodontal ligament stem cells via the miR-7/GDF5/SMAD and p38 MAPK signaling pathway

BackgroundPeriodontal ligament stem cells (PDLSCs) are considered as candidate cells for the regeneration of periodontal and alveolar bone tissues. Antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), which is a newly discovered circular RNA (circRNA), has been reported to act as an miR-7 sponge and to be involved in many biological processes. Here, we investigated the potential roles of CDR1as and miR-7 in the osteogenic differentiation of PDLSCs.MethodsThe expression pattern of CDR1as and miR-7 in PDLSCs during osteogenesis was detected by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Then we overexpressed or knocked down CDR1as or miR-7 to confirm whether they were involved in the regulation of osteoblast differentiation in PDLSCs. Alkaline phosphatase (ALP) and alizarin red S (ARS) staining were used to detect the activity of osteoblasts and mineral deposition. Furthermore, a dual luciferase reporter assay was conducted to analyze the binding of miR-7 to growth differentiation factor (GDF)5. To further verify the role of CDR1as in osteoblast differentiation, we conducted animal experiments in vivo. New bone formation in specimens was analyzed by microcomputed tomography (micro-CT), hematoxylin and eosin staining, and immunofluorescence staining.ResultsWe observed that CDR1as was significantly upregulated during the osteogenic differentiation, whereas miR-7 was significantly downregulated. Moreover, knockdown of CDR1as and overexpression of miR-7 inhibited the ALP activity, ARS staining, and expression of osteogenic genes. Overexpression of miR-7 significantly reduced the activity of luciferase reporter vectors containing the wild-type, but not the mutant, 3’ untranslated region (UTR) sequence of GDF5. Furthermore, knockdown of GDF5 partially reversed the effects of miR-7 inhibitor on osteoblast differentiation. Downregulation of CDR1as or GDF5 subsequently inhibited phosphorylation of Smad1/5/8 and p38 mitogen-activated protein kinases (MAPK), while upregulation of miR-7 decreased the level of phosphorylated Smad1/5/8 and p38 MAPK. In vivo, CDR1as knockdown lead to less bone formation compared with the control group as revealed by micro-CT and the histological analysis.ConclusionsOur results demonstrated that CDR1as acts as a miR-7 inhibitor, triggering the upregulation of GDF5 and subsequent Smad1/5/8 and p38 MAPK phosphorylation to promote osteogenic differentiation of PDLSCs. This study provides a novel understanding of the mechanisms of osteogenic differentiation, and suggests a potential method for promoting bone formation.

Hsa-miR-5582-3P regulatory effect on TGFβ signaling through targeting of TGFβ-R1, TGFβ-R2, SMAD3, and SMAD4 transcripts.

Transforming growth factor β (TGFβ) signaling pathway which is regulated by factors such as microRNAs (miRNAs) has pivotal roles in various cellular processes. Here, we intended to verify bioinformatics predicted regulatory effect of hsa-miR-5582-3P against TGFβ/SMAD signaling pathway components. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) analysis indicated a negative correlation of expression between hsa-miR-5582-3P against TGFβ-R1, TGFβ-R2, SMAD3, and SMAD4 putative target genes in all of tested cell lines. Also, hsa-miR-5582-3P was significantly downregulated in glioma, breast, and ovarian tumor tissues compared with their normal pairs, detected byRT-qPCR. Then dual luciferase assay supported direct interaction between this miRNA and TGFβ-R1, TGFβ-R2, SMAD3, and SMAD4, 3' untranslated region sequences. Western blot analysis confirmed negative effect of hsa-miR-5582-3P overexpression on at least TGFβ-R1 expression. Consistently, hsa-miR-5582-3P overexpression brought about downregulation of TGFβ-R1, TGFβ-R2, SMAD3, and SMAD4 expression in HCT-116 cell line, followed by cell cycle arrest in sub-G1 phase, detected by flow cytometry. Altogether, our data suggest that hsa-miR-5582-3P reduces the TGFβ/SMAD signaling pathway through downregulation of TGFβ-R1, TGFβ-R2, SMAD3, and SMAD4 transcripts. These data introduce hsa-miR-5582-3P as a potential tumor suppressors-miR and a therapy candidate to be tested in cancers in which TGFβ/SMAD is deregulated.

Abstract A65: Dicistronic reporter screen for Internal Ribosome Entry Site (IRES)-mediated translational regulation of truncated p110 ERBB2 isoform

Abstract Background: We and others demonstrated that truncated p110 ERBB2 (p110 t-ERBB2, also known as 611CTF) is a hyperactive truncated ERBB2 isoform capable of increasing cell migration and invasion in multiple cell types in vitro, and induction of human breast epithelial cell (HMLE) xenograft formation in vivo (Ward et al., Oncogene 2013;32(19):2463-74). p110 t-ERBB2 arises through alternative initiation of translation from methionine 611, though the mechanism to account for such cap-independent p110 t-ERBB2 translation is not well studied. mRNA structural elements termed internal ribosome entry sites (IRESs) can initiate translation in a cap-independent manner when canonical cap-dependent translation is severely compromised. It has been previously demonstrated that the human EGFR 5’ untranslated region (UTR) sequence can initiate the expression of a downstream open reading frame via an IRES (Webb TE, et al., Oncogenesis 2015;4:e134). Therefore, we sought to identify presence of a putative IRES within the 5′ UTR that might mediate alternative ERBB2 protein translation initiation under stress conditions and promote p110 t-ERBB2 biosynthesis. Methods: The ERBB2 mRNA 5 ′ UTR from 2 published human ERBB2 transcripts, and several overlapping sequences from full-length ERBB2 start codon to p110 t-ERBB2 start codon were cloned between the Renilla and firefly luciferase open reading frames of pRF and promoter-less vector pRFΔP (SV40 promoter removed by restriction digestion), then the resultant constructs were transiently transfected into different cell lines (BT474, SK-BR3, MCF-7, HeLa, CHO). Three control constructs pRF (empty vector control), pRF-Tub (negative control, containing β-tubulin 5 ′ UTR, which lacks IRES activity), and pRF-myc (positive control, containing the well-characterized c-myc IRES) were parallel-transfected. Luciferase expression was then quantified using a Dual Luciferase Assay Kit (Promega, Madison, WI, USA) following manufacturer’s instructions. Parallel Western blot analysis and qRT-PCR were also conducted. Results: In this report, we demonstrate that in BT474 and SK-BR3 cells, no IRES activity was detected within human ERBB2 5 ′ UTR sequences under nonstressed conditions, or under serum-starvation, hypoxic conditions, or thapsgargin-induced endoplasmic reticulum stress—conditions when global translation was compromised. The construct pRF-+265/+1561 (within ERBB2 mRNA coding sequence, 5 ′ to the p110 t-ERBB2 start codon) displayed a 10-21 fold increase in firefly/Renilla activity when compared with the empty control pRF and negative control pRF-Tub, consistent with the possibility that the region between +265/+1561 may contain a cryptic promoter. Conclusions: These data are inconsistent with the hypothesis that a 5 ′ UTR IRES-mediated mechanism is involved in the translation of p110 t-ERBB2 isoform, and that other mechanisms are operative in alternative translational regulation/biosynthesis of p110 t-ERBB2 isoform. Citation Format: Yu Zong, Yulin Li, Xiaofei Liu, Mark Daniel Pegram. Dicistronic reporter screen for Internal Ribosome Entry Site (IRES)-mediated translational regulation of truncated p110 ERBB2 isoform [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A65.

MicroRNA‑106a regulates the proliferation and invasion of human osteosarcoma cells by targeting VNN2.

MicroRNAs (miRs) serve an essential role in tumorigenesis and are able to act as tumor suppressor genes or oncogenes. miR‑106a has been identified generally as an oncogene in multiple types of human cancer; however, its association with osteosarcoma has not previously been understood. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) was used to detect miR‑106a expression in 18 osteosarcoma tissues compared with paired non‑cancerous adjacent tissues as well as osteosarcoma cell lines (U2OS, Saos‑2 and MG63) compared with a normal osteoblast cell line (hFOB1.19). The biological function of U2OS cells was assessed by using a Transwell cell invasion assay, MTS proliferation assay and flow cytometric analysis following the transfection with lentivirus‑mediated small interfering RNA (miR‑106a‑inhibitor). Western blotting and Luciferase reporters were used to investigate whether VNN2 was a target of miR‑106a in osteosarcoma cells. Based on the RT‑qPCR data, miR‑106a was significantly upregulated in osteosarcoma tissues and osteosarcoma cell lines compared with their control counterparts (P<0.01). The knockdown of miR‑106a resulted in cell proliferation and invasion inhibition. Furthermore, apoptosis enhancement and G2/M cell cycle arrest were detected by flow cytometry. The western blot analysis indicated that U2OS cells infected with miR‑106a‑inhibitor lentivirus had a higher VNN2 protein expression level compared with cells infected with miR‑106a‑negative control lentivirus. Luciferase reporters containing the 3'‑untranslated region sequence of VNN2 messenger RNA demonstrated VNN2 may be a target of miR‑106a. In addition, a negative correlation was confirmed between the expression of VNN2 and miR‑106a in the tumor samples. The results of the present study indicate that the knockdown of miR‑106a overexpressed VNN2 to inhibiting the proliferation, migration and invasion as well as inducing the apoptosis of human osteosarcoma cells.

MiR-122, small RNA annealing and sequence mutations alter the predicted structure of the Hepatitis C virus 5' UTR RNA to stabilize and promote viral RNA accumulation.

Annealing of the liver-specific microRNA, miR-122, to the Hepatitis C virus (HCV) 5′ UTR is required for efficient virus replication. By using siRNAs to pressure escape mutations, 30 replication-competent HCV genomes having nucleotide changes in the conserved 5′ untranslated region (UTR) were identified. In silico analysis predicted that miR-122 annealing induces canonical HCV genomic 5′ UTR RNA folding, and mutant 5′ UTR sequences that promoted miR-122-independent HCV replication favored the formation of the canonical RNA structure, even in the absence of miR-122. Additionally, some mutant viruses adapted to use the siRNA as a miR-122-mimic. We further demonstrate that small RNAs that anneal with perfect complementarity to the 5′ UTR stabilize and promote HCV genome accumulation. Thus, HCV genome stabilization and life-cycle promotion does not require the specific annealing pattern demonstrated for miR-122 nor 5′ end annealing or 3′ overhanging nucleotides. Replication promotion by perfect-match siRNAs was observed in Ago2 knockout cells revealing that other Ago isoforms can support HCV replication. At last, we present a model for miR-122 promotion of the HCV life cycle in which miRNA annealing to the 5′ UTR, in conjunction with any Ago isoform, modifies the 5′ UTR structure to stabilize the viral genome and promote HCV RNA accumulation.

MicroRNA-96 expression induced by low-dose cisplatin or doxorubicin regulates chemosensitivity, cell death and proliferation in gastric cancer SGC7901 cells by targeting FOXO1.

MicroRNA-96 (miR-96) is transcriptionally associated with the induction of chemoresistance following chemotherapy by targeting to FOXO1 mRNA at one of two predicted binding sites in its 3'-untranslated region sequence. The upregulation of miR-96 is associated with a high risk of chemoresistance. Nevertheless, the mechanism by which miR-96 is upregulated remains largely undefined. In the present study, the gastric cancer SGC7901 cell line was treated with different doses of the chemotherapeutic agents cisplatin and doxorubicin. miR-96 expression was analyzed by reverse transcription-quantitative polymerase chain reaction at different time points. Western blot and chromatin immunoprecipitation were performed to analyze the expression levels of the target gene. The effects of miR-96 on chemosensitivity were assessed by a carboxyfluorescein succinimidyl ester/propidium iodide labeling assay, and its effects on proliferation were assessed by Cell Counting Kit-8 or EdU staining assays. The results demonstrated that treatment with a low dose of either chemotherapeutic agent induced miR-96 expression. Upregulation of miR-96 caused the post-transcriptional repression of FOXO1 expression. Decreases in FOXO1 protein levels led to a decrease in the transcriptional activity of the cyclin-dependent kinase inhibitor 1A (CDKN1A, also known as p21) promoter region, and thus the expression of p21 was downregulated in a tumor protein p53-independent manner. As a result, induction of miR-96 expression caused chemoresistance and promoted proliferation in SGC7901 cells. Taken together, the results of the present study revealed that treatment with cisplatin or doxorubicin could induce expression of miR-96 at certain doses. Upregulation of miR-96 is partially associated with chemoresistance and miR-96 can also promote cell proliferation by repressing p21.